Light-gated integrator for highlighting kinase activity in living cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1138651 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29912142_1.fastq.gz | Fastqsanger.gz | |||
| SRR29912142_2.fastq.gz | Fastqsanger.gz | |||
| SRR29912143_1.fastq.gz | Fastqsanger.gz | |||
| SRR29912143_2.fastq.gz | Fastqsanger.gz | |||
| SRR29912144_1.fastq.gz | Fastqsanger.gz |
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Project description:Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into “permanent” fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.
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Project description:Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into "permanent" fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA signaling heterogeneity in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.
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Project description:Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration Targeting-Sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3’ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disrupting histone mRNA processing, and leading to polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites. HITseq was used to interrogate chromatin binding sites of of proteins in the complex (including INIP, INTS3, INTS9, INTS11, NABP1, NABP2, NELFA, NELFB, NELFCD, NELFE, SPT5). These proteins were fused to LEDGF-IBD and expressed in mouse LEDGF-/-MEF cells. The fusion proteins then target HIV integration to the chromatin binding sites. The HIV integration sites were identified using linker-mediated PCR and highthroughput sequencing and serve as surrogate for chromatin binding sites. Mapping was done using mouse genome mm9.
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The incorporation of light-responsive domains into engineered proteins has produced optogenetic tools with the ability to regulate protein localization, interactions, and function with light. We introduced this mode of regulation into proximity labeling (PL), which has been a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through a combination of structure-guided screening and yeast display directed evolution, we inserted the light sensitive LOV domain into a surface exposed loop of the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. We showed that "LOV-Turbo" works in multiple organelles and cell types and can dramatically reduce background labeling in biotin-rich environments such as living neurons. We utilized LOV-Turbo's reversibility to perform pulse-chase labeling followed by organelle fractionation to discover endogenous proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. Lastly, we showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from proximal luciferase in living cells, opening the door to chemical and/or interaction dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL and expands the scope of experimental questions that can be addressed using PL.
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Project description:MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The mechanistic steps of miRNA biogenesis are well understood at molecular level1,2. Subsequent miRNA duplex loading into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing3-5. The Integrator complex has been previously identified as important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination, processes dependent on Integrator’s catalytical activity6-9. Here we report that absence of the Integrator complex leads to a global loss of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Indeed, Integrator enhances miRNA-Ago2 interaction in vitro, which functionally translates to amplified miRNA target cleavage. These findings demonstrate for the first time cytoplasmic Integrator complex functions and stress an indirect role of Integrator in transcription regulation through modulation of miRNA abundance and stability.
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Project description:Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, we performed high-throughput RNAi screening of the Drosophila Metallothionein A (MtnA) promoter. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress. Integrator complex subunit 11 (IntS11) endonucleolytically cleaves MtnA transcripts, resulting in premature transcription termination and degradation of the nascent RNAs by the RNA exosome, a complex also identified in the screen. Using RNA-seq, we then identified >400 additional Drosophila protein-coding genes whose expression increases upon Integrator depletion. We focused on a subset of these genes and confirmed that Integrator is bound to their 5' ends and negatively regulates their transcription via IntS11 endonuclease activity. Many non-catalytic Integrator subunits, which are largely dispensable for snRNA processing, also have regulatory roles at these protein-coding genes, possibly by controlling Integrator recruitment or RNA polymerase II dynamics. Altogether, our results suggest that attenuation via Integrator cleavage limits production of many full-length mRNAs, allowing precise control of transcription outputs.
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